Odcase inhibitors as anti-virals and antibiotics

ABSTRACT

The present invention includes the utility of anti-viral and/or antibacterial effective amounts of 6-substituted nucleoside derivatives of formula (I) (e.g. 6-iodouridine and 6-iodouridine monophosphate) in the treatment or prevention of viral infections (e.g. Flavivridae, Bunyaviridae, or Togaviridae, or viral infections of hepatitis C, hepatitis B, herpes, influenza, HIV, polio, Coxsackie A/B, rhino, small pox, Ebola, West Nile, or corona virus) and/or bacterial infections (e.g.  H. pylori, S. Aureus, B. anthracis, Mycobacterial tuberculosis, M. leprae, M. avium, P. aueruginosa, Streptococcal  species, and  Pneumocystis carinii ).

FIELD OF THE INVENTION

The present invention relates to methods of using certain 6-substituteduridine compounds for the treatment and prevention of viral andbacterial infections.

BACKGROUND OF THE INVENTION

ODCase (EC 4.1.1.23) plays a central role in the de novo synthesis ofuridine-5′-O-monophosphate (UMP). UMP is a building block, synthesizedde novo from aspartic acid, for the synthesis of other pyrimidinenucleotides such as uridine-5′-O-triphosphate (UTP),cytidine-5′-O-triphosphate (CTP), thymidine-5′-O-triphosphate (TMP) and2′-deoxy-cytidine-5′-O-triphosphate (dCTP) (FIG. 1). Pyrimidinenucleotides are the building blocks for the synthesis of RNA and DNA,the essential molecules for cell replication and survival. Due to itsimportant role in the cell's de novo nucleic acid synthesis, ODCase ispresent in bacteria, archea, parasites and in humans, i.e. almost everyspecies except in viruses. This enzyme catalyzes the decarboxylation oforotidine monophosphate (OMP) to uridine monophosphate (UMP) (compounds1 and 2 in the final step in FIG. 1). This enzyme is particularlyinteresting for enzymologists because it exhibits an extraordinary levelof catalytic rate enhancement of over 17 orders of magnitude compared tothe uncatalyzed decarboxylation reaction in water at neutral pH 7.0, 25°C.^(i,ii) An uncatalyzed decarboxylation of OMP takes about 78 millionyears, and the enzymic decarboxylation occurs at a millisecond timescale. Thus, ODCase is one of the most proficient members of the enzymicworld.^(ii,iii,iv,v)

Interestingly, decarboxylases found in Nature use either a cofactor orcovalent intermediates during the catalysis of decarboxylationreactions.^(vi,vii) For example, thiamin diphosphate-dependent, indolepyruvate decarboxylase (IPDC) uses thiamin as a cofactor and there arecovalent intermediates formed with the cofactor during thedecarboxylation process. ODCase is thought to be quite unusual incatalyzing decarboxylation with such proficiency without the help of anyco-factors, metals, or covalent-intermediates.^(i,ii,iii) Oneinteresting difference when one looks at this enzyme across species isthat in certain higher level organisms such as human or mouse, ODCase isa part of the bifunctional enzyme, UMP synthase.^(viii) In pathogenicorganisms such as bacteria, fungi and parasites, ODCase is amonofunctional enzyme.^(ix,x) In all species, ODCase (whethermonofunctional or bifunctional) is active as a dimeric unit.

In general, investigations targeting ODCase focused on malaria, cancerand few antiviral investigations. In the past two decades, severalanalogs of OMP were investigated extensively to understand the catalyticmechanism of ODCase.^(iii,xi) Among these analogs, 6-aza-UMP (3) and6-hydroxy-UMP (or BMP, 4), pyrazofurin, xanthosine-5′-monophosphate(XMP, 12) and 6-thiocarboxamido-UMP (13) are some of the potentinhibitors that were studied against ODCase (FIG. 2).^(xii,xiii,xiv)However, the development of inhibitor candidates has been limited due totheir toxicities and lack of specificity.^(xii) There is also verylimited or non-existent structure-activity relationship investigationsand inhibitor design against ODCase. Thus, ODCase has not gained muchtraction in 1980s and 1990s as a drug target.

Aside from its obvious pharmacological interest, ODCase has been afavorite enzyme for biochemists and structural biologists due to itsunusual catalytic properties. A number of mechanisms were proposed priorto and after the availability of X-ray crystal structures for severalODCases in 2000.^(xv,xvi,xvii,xviii) Although ideas of covalentcatalysis were discussed, none of the mechanisms presented included acovalent species formation as a key step during the decarboxylation byODCase. An analysis of the catalytic site of ODCase fromMethanobacterium thermoautotrophicum (Mt) revealed two aspartateresidues (Asp70 and Asp75B, the latter contributed by the second subunitof the dimeric ODCase) and two lysine residues (Lys42 and Lys72) thatare held via a strong network of hydrogen bonds (FIG. 3). Analyses ofseveral co-crystal structures of ODCase with a variety of ligandsconfirm that these residues are held tightly in their respectivepositions in the active site and there is less than 0.5 Å movement inthe positions of the side chains of these residues. Existing evidencedoes not support any active site residue forming a covalent bond eitherto the substrate during catalysis or to any knowninhibitor.^(iii,xvi,xix,xx,xxi) The above four residues are proposed toexert strong steric and electrostatic stress onto the C-6 carboxylategroup of OMP and eliminate the carboxyl group.^(xvi)

The x-ray crystal structures of ODCase from ten different species areknown today. In 2000, four x-ray crystal structures of ODCase broughtinsights into the catalytic mechanism of this enzyme. Based on thestructure of S. cerevisiae ODCase complexed with the transition-stateanalogue BMP (4), a transition-state stabilization mechanism of OMPdecarboxylation was proposed.^(xviii) A similar proposal was alsosuggested by Appleby et al. based on the crystal structure of ODCase(Bacillus subtilis) complexed with the product, UMP.^(xvii) Theseauthors suggested that the decarboxylation reaction proceeds via anelectrophilic substitution in which C-6 is protonated by Lys62 as thecarbon dioxide molecule is released.^(xvii) The structure of the ODCaseenzyme from E. coli co-crystallized with BMP was the basis of theproposal submitted by Harris et al.^(xxii) Based on the proximity of thecarboxylate moiety on OMP (1) and Asp71 residue in the active site ofODCase, it was proposed that OMP decarboxylation depends on theexistence of a shared proton between Asp71 and the carboxyl group of thesubstrate.^(xxii) A similar mechanism involving electrostatic repulsionwas put forward by Wu et al.^(xvi) This mechanism of OMP decarboxylationis based on the principles of the Circe effect described by Jencks in1975.^(xxiii) The Circe effect states that only the reactive group ofthe substrate needs to be destabilized. The strong interaction betweenthe unreactive part of the substrate and the enzyme active site providesthe energy to directly destabilize the reactive group of thesubstrate.^(xxiii) The electrostatic repulsion mechanism points to theactive site aspartate residue. In four different species the locationand function of this residue is highly conserved. The catalyticresidues, Asp70 and Lys72 are located near the reaction center C-6 ofthe pyrimidine ring of the substrate OMP and Asp70 (M.thermoautotrophicum) was postulated to provide the electrostaticdestabilization of the enzyme-substrate complex. Lys72 in the activesite furnishes the proton to neutralize the carbanion developed afterthe departure of the carboxylate.^(xvi) Despite several x-ray structuresand in-depth enzymology in the past two decades, ODCase continues tochallenge biochemists with still-unresolved mechanism and new twists(vide infra).

In the active site of ODCase, the monophosphate group of OMP is proposedto bind first and this group contributes the largest energy required forthe binding of the substrate to ODCase.^(xxiv) The removal of phosphatefrom the molecule of substrate resulted in a significantly lowercatalytic efficiency measured as the second-order rate constant(k_(cat)/K_(M)) for the catalysis of substrate to product.^(xxiv) In aninteresting experiment, the binding of phosphite dianion (HPO₃ ²⁻) toODCase (from S. cerevisiae) resulted in an 80,000 fold increase in thesecond order rate constant for the decarboxylation of the truncatedsubstrate lacking a phosphate moiety.^(xxv) Thus, the phosphate group isan important component for ODCase binding. Thus, in order for nucleosidedrugs (correct terminology is prodrugs) to be active against ODCase invivo, the nucleoside compound has to be converted into its monophosphateform inside the cell by any nucleoside kinase and then inhibit ODCase(whether in a pathogen or human cell). This is very similar to othernucleoside drugs such as AZT, 3TC, gemcitabine among several nucleosidedrugs that are clinically used, thus there is a good possibility for the“nucleoside forms” of ODCase inhibitors to function as drugs.

The market size for antimicrobial agents is more than US$25 billion peryear and the emergence of bacterial resistance worldwide is a limitingfactor to current drugs. In the US alone, more than 80 million peopleare infected with H. pylori. Worldwide, 25-75% of people are infectedwith H. pylori depending on the region and will eventually developgastritic diseases, many leading to cancer. Staphylococcus aureus hasbecome a serious issue in hospitals and has recently become acommunity-based infection with resistance against the most effectivedrugs, including the widely-used methicillin, and the drug of lastresort, vancomycin. New and effective drugs against these two infectiousdiseases, especially against the new ODCase target, have the potentialto achieve significant market penetration.

The cellular machinery for the synthesis of pyrimidine nucleotidesvaries in different bacterial species. For example, an analysis of thecomplete genome of H. pylori reveals the metabolic pathways for purineand pyrimidine biosynthesis.^(xxvi,xxvii) Interestingly, there are noidentifiable genes for the enzymes in the pyrimidine salvage pathway(except one) in H. pylori genome and thus, this organism is completelydependent on the de novo synthesis of pyrimidinenucleotides.^(xxvii,xxviii) This clearly suggests that inhibition ofODCase will have a fatal effect on the survival of H. pylori. Thisrationale provides a strong basis to design inhibitors against ODCase,as antibacterial agents. S. aureus based on its complete genome analysis(strain MRSA A252), appears to lack five of the eight enzymes in thepyrimidine salvage pathway.

ODCase has also been identified as a target for drugs directed againstRNA viruses like pox and flavi viruses; the former causing increasingconcern as a potential bio-terrorist weapon.^(xxix,xxx,xxxi,xxxii)ODCase inhibitors have also been effective against West Nile virus, arecent threat to humans and birds in the US and Canada.^(xxxiii)

There remains a need for new inhibitors of ODCase as therapeutic agents,for example, for the prevention and treatment of viral and bacterialinfections.

SUMMARY OF THE INVENTION

Several C6 derivatives of uridine were prepared and found to benoncovalent (competitive) and covalent (irreversible) inhibitors againstODCase. These compounds also exhibited potent anti-viral and antibioticactivity.

Accordingly, the present invention includes a method of treating orpreventing viral and/or bacterial infections comprising administering toa subject in need thereof a an anti-viral effective amount and/or anantibacterial effective amount of a compound selected from a compound ofFormula I, tautomers thereof and pharmaceutically acceptable salts,solvates, and prodrugs thereof:

wherein,

-   R¹ is selected from I, Br, C₁, N₃ and NO₂;-   R² is selected from H, halo, C₁-C₆alkyl, C₁-C₆alkoxy,    fluoro-substituted-C₁-C₆alkyl, fluoro-substituted-C₁-C₆alkoxy, N₃,    NH₂ and CN;-   R³ is selected from OH, NH₂, H and NHC(O)C₁-C₆alkyl;-   Z is selected from:

wherein,

-   R⁴ is selected from H, C_(j)—C₆alkyl and    hydroxy-substituted-C₁-C₆alkyl;-   One of R⁵ and R⁶ is hydrogen and the other is selected from H, OH    and F and one of R^(5′) and R^(6′) is hydrogen and the other is    selected from H, OH and F or R⁵ and R⁶ or R^(5′) and R^(6′) together    may be ═O or ═CH₂;-   R⁷ is selected from H, F and OH;-   R⁸ is selected from H, C(O)C₁-C₆alkyl, P(O)(OH)₂, P(O)(OC₁-C₆alkyl)₂    and P(O)(OC₁-C₆alkyl)OH;-   R⁹ is selected from H, N₃, CN, C₁-C₆alkyl; and-   X is selected from —CH₂—O—, O—CH₂— and —S—CH₂—.

In further embodiments, the present invention includes a use of acompound selected from a compound of Formula I as defined above, andpharmaceutically acceptable salts, solvates, and prodrugs thereof, forthe prevention or treatment of viral infections and/or bacterialinfections, as well as a use of a compound selected from a compound ofFormula I as defined above, and pharmaceutically acceptable salts,solvates, and prodrugs thereof, for the preparation of a medicament forthe prevention or treatment of viral infections and/or bacterialinfections.

The present invention further includes a method of preventing ortreating an infection of one or more bacteria and/or one or more viruesin a subject comprising administering to the subject an anti-viraleffective amount and/or an antibacterial effective amount of a compoundselected from a compound of Formula I as defined above, andpharmaceutically acceptable salts, solvates, and prodrugs thereof.

The present invention also includes a use of a compound of Formula I asdefined above, and pharmaceutically acceptable salts, solvates, andprodrugs thereof, to prevent or treat an infection of a virus and/orbacterium in a subject as well as a use of a compound of Formula I asdefined above, and pharmaceutically acceptable salts, solvates, andprodrugs thereof, to prepare a medicament to prevent or treat aninfection of a virus and/or bacterium in a subject

According to another aspect of the present invention, there is includeda pharmaceutical composition for the treatment or prevention of viralinfections and/or bacterial infections comprising a anti-viral effectiveamount and/or a antibacterial effective amount of a compound selectedfrom a compound of Formula I as defined above, and pharmaceuticallyacceptable salts, solvates, and prodrugs thereof, and a pharmaceuticallyacceptable carrier therefore.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will know be described in great detail with reference tothe drawings in which:

FIG. 1 is a schematic showing the de novo synthesis of uridinemonophosphate from aspartic acid (prior art).

FIG. 2 is a schematic showing the chemical structures of analogs oforotidine monophosphate (OMP) that are known as inhibitors of ODCase.

FIG. 3 shows the X-ray structure of the catalytic site of ODCase fromMethanobacterium thermoautotrophicum.

FIG. 4 shows bargraphs of the results of an antiviral assay in MDCK celllines against influenza A/WSN/33. Panels A and B reflect the results oftwo separate experiments. Numbers on top of each bar represent the %protection due to the inhibitor in comparison to the control.

FIG. 5 shows a bargraph of the results of an antiviral assay in L2 celllikes against MHV-1 (mouse SARS-like CoV).

FIG. 6 shows graphs of primary cell toxicity data using representativecompounds of Formula I.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

The term “C_(1-n)alkyl” as used herein means straight and/or branchedchain, saturated alkyl radicals containing from one to “n” carbon atomsand includes (depending on the identity of n) methyl, ethyl, propyl,isopropyl, n-butyl, s-butyl, isobutyl, t-butyl, 2,2-dimethylbutyl,n-pentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, n-hexyl andthe like, where the variable n is an integer representing the largestnumber of carbon atoms in the alkyl radical.

The term “fluoro-substituted C_(1-n)alkyl” as used herein means straightand/or branched chain, saturated alkyl radicals containing from one to ncarbon atoms in which one or all of the hydrogen atoms have beenreplaced with a fluorine, and includes (depending on the identity of“n”) trifluoromethyl, pentafluoroethyl, fluoromethyl and the like, wherethe variable n is an integer representing the largest number of carbonatoms in the alkyl radical.

The term “hydroxy-substituted C_(1-n)alkyl” as used herein meansstraight and/or branched chain, saturated alkyl radicals containing fromone to n carbon atoms in which one or two of the hydrogen atoms havebeen replaced with a hydroxyl group, and includes (depending on theidentity of “n”) CH₂OH, CHOHCH₂CH₃, CH₂CHOHCH₂CH₂OH and the like, wherethe variable n is an integer representing the largest number of carbonatoms in the alkyl radical.

The term “halo” as used herein means halogen and includes chloro,fluoro, bromo and iodo.

The term “tautomer” as used herein refers to compounds that areinterconvertible by a formal migration of a hydrogen atom or proton,accompanied by a switch of a single bond and an adjacent double bond. Insolutions where tautomerization is possible, a chemical equilibrium ofthe tautomers will be reached. The exact ratio of the tautomers dependson several factors, including temperature, solvent and pH.

The term “solvate” as used herein means a compound of Formula I, or asalt of a compound of Formula I, wherein molecules of a suitable solventare incorporated in the crystal lattice. A suitable solvent isphysiologically tolerable at the dosage administered. Examples ofsuitable solvents are ethanol, water and the like. When water is thesolvent, the molecule is referred to as a “hydrate”.

The term “compound(s) of the invention” as used herein means compound(s)of Formula I, and salts, solvates and prodrugs thereof.

The term “pharmaceutically acceptable salt” means an acid addition saltor a basic addition salt which is suitable for or compatible with thetreatment of patients.

The term “pharmaceutically acceptable acid addition salt” as used hereinmeans any non-toxic organic or inorganic salt of any base compound ofthe invention, or any of its intermediates. Basic compounds of theinvention that may form an acid addition salt include, for example,where the R² and/or R³ is NH₂ and NHC₁₋₆alkyl. Illustrative inorganicacids which form suitable salts include hydrochloric, hydrobromic,sulfuric and phosphoric acids, as well as metal salts such as sodiummonohydrogen orthophosphate and potassium hydrogen sulfate. Illustrativeorganic acids that form suitable salts include mono-, di-, andtricarboxylic acids such as glycolic, lactic, pyruvic, malonic,succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic,benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonicacids such as p-toluene sulfonic and methanesulfonic acids. Either themono or di-acid salts can be formed, and such salts may exist in eithera hydrated, solvated or substantially anhydrous form. In general, theacid addition salts of the compounds of the invention are more solublein water and various hydrophilic organic solvents, and generallydemonstrate higher melting points in comparison to their free baseforms. The selection of the appropriate salt will be known to oneskilled in the art. Other non-pharmaceutically acceptable acid additionsalts, e.g. oxalates, may be used, for example, in the isolation of thecompounds of the invention, for laboratory use, or for subsequentconversion to a pharmaceutically acceptable acid addition salt.

The term “pharmaceutically acceptable basic addition salt” as usedherein means any non-toxic organic or inorganic base addition salt ofany acid compound of the invention, or any of its intermediates. Acidiccompounds of the invention that may form a basic addition salt include,for example, where R⁸ is a phosphate. Illustrative inorganic bases whichform suitable salts include lithium, sodium, potassium, calcium,magnesium or barium hydroxide. Illustrative organic bases which formsuitable salts include aliphatic, alicyclic or aromatic organic aminessuch as methylamine, trimethylamine and picoline, alkylammonias orammonia. The selection of the appropriate salt will be known to a personskilled in the art. Other non-pharmaceutically acceptable basic additionsalts, may be used, for example, in the isolation of the compounds ofthe invention, for laboratory use, or for subsequent conversion to apharmaceutically acceptable acid addition salt.

The term “viral infection” as used herein refers to the detrimentalcolonization of a host organism by one or more foreign viruses. Theinfecting virus seeks to utilize the host's resources in order tomultiply (usually at the expense of the host) and interferes with thenormal functioning of the host and can lead to chronic wounds, gangrene,loss of an infected limb, disease and even death. Examples of viralinfections include, but are not limited to, RNA viral infections,including infections by the following virus families: Flaviviridae(yellow fever [YF], Dengue type 4 and Japanese encephalitis [JE]viruses), Bunyaviridae (Punta Toro [PT] and sandfly fever [SF] viruses)and Togaviridae (Venezuelan equine encephalomyelitis [VEE] virus), aswell as hepatitis C virus (HCV), hepatitis B virus (HBV), herpesviruses, influenza virus, human immuno virus (HIV), polio virus,Coxsackie A and B viruses, Rhino virus, small pox virus, Ebola virus,West Nile virus and coronavirus (such as severe acute respiratorysyndrome or SARS).

The term “bacterial infection” as used herein refers to the detrimentalcolonization of a host organism by one or more foreign bacteria. Theinfecting bacteria seeks to utilize the host's resources in order tomultiply (usually at the expense of the host) and interferes with thenormal functioning of the host and can lead to chronic wounds, gangrene,loss of an infected limb, disease and even death. Examples of bacterialinfections include all bacterial species in which ODCase is an importantcomponent of pyrimidine synthetic pathway, for example, but are notlimited to, H. pylori, S. aureus, B. anthracis, Mycobacterial speciessuch as M. tuberculosis, M. leprae, M. avium, P. aueruginosa,Streptococcal species, Pneumocystis carinii and the like.

The term a “therapeutically effective amount”, “effective amount” or a“sufficient amount” of a compound of the present invention is a quantitysufficient to, when administered to the subject, including a mammal, forexample a human, effect beneficial or desired results, includingclinical results, and, as such, an “effective amount” or synonym theretodepends upon the context in which it is being applied. For example, inthe context of inhibiting ODCase, for example, it is an amount of thecompound sufficient to achieve such an inhibition in ODCase activity ascompared to the response obtained without administration of thecompound. In the context of disease, therapeutically effective amountsof the compounds of the present invention are used to treat, modulate,attenuate, reverse, or effect bacterial and/or viral infections in amammal. An “effective amount” is intended to mean that amount of acompound that is sufficient to treat, prevent or inhibit viral and/orbacterial infection or a disease associated with viral and/or bacterialinfection. In some suitable embodiments, viral and/or bacterialinfection or the disease or disorder associated with viral and/orbacterial infection is caused by an RNA virus (as defined above) and/orbacteria, thus it is the amount sufficient to, when administered to thesubject, including a mammal, e.g., a human, to treat, prevent or inhibitviral and/or bacterial infection or a disorder associated with viraland/or bacterial infection. The amount of a given compound of thepresent invention that will correspond to such an amount will varydepending upon various factors, such as the given drug or compound, thepharmaceutical formulation, the route of administration, the type ofdisease or disorder, the identity of the subject or host being treated,and the like, but can nevertheless be routinely determined by oneskilled in the art. Also, as used herein, a “therapeutically effectiveamount” of a compound of the present invention is an amount whichprevents, inhibits, suppresses or reduces viral and/or bacterialinfection (e.g., as determined by clinical symptoms or the amount ofvirues and/or bacteria) in a subject as compared to a control. Asdefined herein, a therapeutically effective amount of a compound of thepresent invention may be readily determined by one of ordinary skill byroutine methods known in the art.

In an embodiment, a therapeutically effective amount of a compound ofthe present invention ranges from about 0.1 to about 7 mg/kg bodyweight, suitably about 1 to about 5 mg/kg body weight, and moresuitably, from about 2 to about 3 mg/kg body weight. The skilled artisanwill appreciate that certain factors may influence the dosage requiredto effectively treat a subject, or prevent a subject, from beingafflicted with viral and/or bacterial infections and these factorsinclude, but are not limited to, the severity of the disease ordisorder, previous treatments, the general health and/or age of thesubject and other diseases present.

Moreover, a “treatment” or “prevention” regime of a subject with atherapeutically effective amount of the compound of the presentinvention may consist of a single administration, or alternativelycomprise a series of applications. For example, the compound of thepresent invention may be administered at least once a week. However, inanother embodiment, the compound may be administered to the patient fromabout one time per week to about once daily for a given treatment. Thelength of the treatment period depends on a variety of factors, such asthe severity of the disease, the age of the patient, the concentrationand the activity of the compounds of the present invention, or acombination thereof. It will also be appreciated that the effectivedosage of the compound used for the treatment or prophylaxis mayincrease or decrease over the course of a particular treatment orprophylaxis regime. Changes in dosage may result and become apparent bystandard diagnostic assays known in the art. In some instances, chronicadministration may be required. The compounds of the present inventionmay be administered before, during or after exposure to a viral and/orbacterial infection.

As used herein, “administered contemporaneously” means that twosubstances are administered to a subject such that they are bothbiologically active in the subject at the same time. The exact detailsof the administration will depend on the pharmacokinetics of the twosubstances in the presence of each other, and can include administeringone substance within 24 hours of administration of the other, if thepharmacokinetics are suitable. Design of suitable dosing regimens areroutine for one skilled in the art. In particular embodiments, twosubstances will be administered substantially simultaneously, i.e.within minutes of each other, or in a single composition that comprisesboth substances.

As used herein, and as well understood in the art, “treatment” is anapproach for obtaining beneficial or desired results, including clinicalresults. Beneficial or desired clinical results can include, but are notlimited to, alleviation or amelioration of one or more symptoms orconditions, diminishment of extent of disease, stabilized (i.e. notworsening) state of disease, preventing spread of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable. “Treatment” can also mean prolongingsurvival as compared to expected survival if not receiving treatment.

“Palliating” a disease or disorder means that the extent and/orundesirable clinical manifestations of a disorder or a disease state arelessened and/or time course of the progression is slowed or lengthened,as compared to not treating the disorder.

The term “prevention” or “prophylaxis”, or synonym thereto, as usedherein refers to a reduction in the risk or probability of a patientbecoming afflicted with a viral and/or bacterial infection ormanifesting a symptom associated with a viral and/or bacterialinfection.

To “inhibit” or “suppress” or “reduce” a function or activity, such asODCase activity, is to reduce the function or activity when compared tootherwise same conditions except for a condition or parameter ofinterest, or alternatively, as compared to another conditions.

The term “subject” or “patient” or synonym thereto, as used hereinincludes all members of the animal kingdom, especially mammals,including human. The subject or patient is suitably a human.

II. Methods of the Invention

The present invention includes a method of treating or preventing viralinfections and/or bacterial infections comprising administering to asubject in need thereof an anti-viral effective and/or an antibacterialeffective amount compound selected from a compound of Formula I,tautomers thereof and pharmaceutically acceptable salts, solvates, andprodrugs thereof:

wherein,

-   R¹ is selected from I, Br, C₁, N₃ and NO₂;-   R² is selected from H, halo, C₁-C₆alkyl, C₁-C₆alkoxy,    fluoro-substituted-C₁-C₆alkyl, fluoro-substituted-C₁-C₆alkoxy, N₃,    NH₂ and CN;-   R³ is selected from OH, NH₂, H and NHC(O)C₁-C₆alkyl;-   Z is selected from:

wherein,

-   R⁴ is selected from H, C₁-C₆alkyl and    hydroxy-substituted-C₁-C₆alkyl;-   One of R⁵ and R⁶ is hydrogen and the other is selected from H, OH    and F and one of R^(5′) and R^(6′) is hydrogen and the other is    selected from H, OH and F or R⁵ and R⁶ or R^(5′) and R^(6′) together    may be ═O or ═CH₂;-   R⁷ is selected from H, F and OH;-   R⁸ is selected from H, C(O)C₁-C₆alkyl, P(O)(OH)₂, P(O)(OC₁-C₆alkyl)₂    and P(O)(OC₁-C₆alkyl)OH;-   R⁹ is selected from H, N₃, CN, C₁-C₆alkyl; and-   X—Y is selected from —CH₂—O—, —O—CH₂— and —S—CH₂—.

In the method of the invention, R¹ in the compounds of Formula I isselected from I, Br, C₁, N₃ and NO₂. In embodiments of the invention, R¹in the compounds of Formula I is selected from I, Br or Cl. NO₂,N(C₁-C₄alkyl)₂, NHC(O)C₁-C₄alkyl and NHC(O)OC₁-C₄alkyl. In furtherembodiments of the invention, R¹ in the compounds of Formula I is I.

In the method of the invention, R² in the compounds of Formula I isselected H, halo, C₁-C₆alkyl, C₁-C₆alkoxy,fluoro-substituted-C₁-C₆alkyl, fluoro-substituted-C₁-C₆alkoxy, N₃, NH₂and CN. In embodiments of the invention, R² in the compounds of FormulaI is H. In further embodiments of the invention, R² in the compounds ofFormula I is halo, suitably F, Br or I, more suitably F, when the methodis for the treatment or prevention of bacterial infections.

In the method of the invention, R³ in the compounds of Formula I isselected from OH, NH₂, H and NHC(O)C₁-C₆alkyl. In embodiments of theinvention, R³ in the compounds of Formula I is selected OH and NH₂. WhenR³ in the compounds of Formula I is selected OH and NH₂, the compoundsof formula I may exist as one of the following tautomers:

where W is O or NH. In embodiments of the invention W is O and thefavoured tautomer is:

In the method of the invention, Z in the compounds of Formula I isselected from:

In an embodiment of the invention, Z is of the Formula II.

In the method of the invention, R⁴ in the compounds of Formula I isselected from H, C₁-C₆alkyl and hydroxy-substituted-C₁-C₆alkyl. In anembodiment of the invention R⁴ in the compounds of Formula I is H.

In the method of the invention, the compounds of Formula I include thosein which one of R⁵ and R⁶ is hydrogen and the other is selected from H,OH and F and one of R^(5′) and R^(6′) is hydrogen and the other isselected from H, OH and F or R⁵ and R⁶ or R^(5′) and R^(6′) together maybe ═O or ═CH₂. In an embodiment of the invention, R⁵ and R^(5′) are bothOH and R⁶ and R^(6′) are both H. In a further embodiment of theinvention, R⁵ is H, R^(5′) is OH and R⁶ and R^(6′) are both H.

In the method of the invention, R⁷ in the compounds of Formula I isselected from H, F and OH, suitably H or OH.

In the method of the invention, R⁸ in the compounds of Formula I isselected from H, C(O)C₁-C₆alkyl, P(O)(OH)₂, P(O)(OC₁-C₆alkyl)₂ andP(O)(OC₁-C₆alkyl)OH. In embodiments of the invention, R⁸ in thecompounds of Formula I is selected from H, C(O)C₁-C₄alkyl, P(O)(OH)₂,P(O)(OC₁-C₄alkyl)₂ and P(O)(OC₁-C₄alkyl)OH. In further embodiments ofthe invention, R⁸ in the compounds of Formula I is selected from H,C(O)CH₃, P(O)(OH)₂, P(O)(OCH₃)₂ and P(O)(OCH₃)OH. In still furtherembodiments of the invention, R⁸ in the compounds of Formula I isselected from H, C(O)CH₃, and P(O)(OH)₂.

In the method of the invention, R⁹ in the compounds of Formula I isselected from H, N₃, CN, C₁-C₆alkyl. Suitably R⁹ is H.

In the method of the invention, X—Y in the compounds of Formula I isselected from —CH₂—O—, —O—CH₂— and —S—CH₂—. Suitably X—Y is —O—CH₂—.

It is an embodiment of the invention that R³ is OH and Z is Formula II.In these compounds the keto tautomeric form is preferred. Accordingly,it is an embodiment of the invention that the compound of Formula I inthe method of the invention has the following structure.

In specific embodiments of the invention, the compound of Formula I inthe method of the invention for treating or preventing bacterialinfections is selected from:

-   6-iodo uridine;-   5-fluoro-6-iodo uridine;-   6-iodo uridine-5′-O-monophosphate;-   5-fluoro-6-iodo uridine-5′-O-monophosphate;-   6-iodo uridine 5′-acetate;-   5-fluoro-6-iodo uridine-5′-acetate;-   6-iodo 2′-deoxyuridine;-   5-fluoro-6-iodo 2′-deoxyuridine;-   6-iodo 2′-deoxyuridine-5′-O-monophosphate;-   5-fluoro-6-iodo 2′-deoxyuridine-5′-O-monophosphate, and    pharmaceutically acceptable salts, solvates, and prodrugs thereof.

In other embodiments of the invention, the compound of Formula I in themethod of the invention for the treatment or prevention of viralinfections is selected from:

-   6-iodo uridine;-   6-iodo uridine-5′-O-monophosphate;-   6-iodo uridine 5′-acetate;-   6-iodo 2′-deoxyuridine;-   6-iodo 2′-deoxyuridine-5′-O-monophosphate, and    pharmaceutically acceptable salts, solvates, and prodrugs thereof.

The present invention includes a method of treating or preventing viralinfections comprising administering to a subject in need thereof ananti-viral effective amount compound selected from a compound of FormulaI, tautomers thereof and pharmaceutically acceptable salts, solvates,and prodrugs thereof:

wherein,

-   R¹ is selected from I, Br, C₁, N₃ and NO₂;-   R² is H;-   R³ is selected from OH, NH₂, H and NHC(O)C₁-C₆alkyl;-   Z is selected from:

wherein,

-   R⁴ is selected from H, C₁-C₆alkyl and    hydroxy-substituted-C₁-C₆alkyl;-   One of R⁵ and R⁶ is hydrogen and the other is selected from H, OH    and F and one of R^(5′) and R^(6′) is hydrogen and the other is    selected from H, OH and F or R⁵ and R⁶ or R^(5′) and R^(6′) together    may be ═O or ═CH₂;-   R⁷ is selected from H, F and OH;-   R⁸ is selected from H, C(O)C₁-C₆alkyl, P(O)(OH)₂, P(O)(OC₁-C₆alkyl)₂    and P(O)(OC₁-C₆alkyl)OH;-   R⁹ is selected from H, N₃, CN, C₁-C₆alkyl; and-   X—Y is selected from —CH₂—O—, —O—CH₂— and —S—CH₂—.

The present invention includes a method of treating or preventingbacterial infections comprising administering to a subject in needthereof an antibacterial effective amount compound selected from acompound of Formula I, tautomers thereof and pharmaceutically acceptablesalts, solvates, and prodrugs thereof:

wherein,

-   R¹ is selected from I, Br, C₁, N₃ and NO₂;-   R² is selected from H, halo, C₁-C₆alkyl, C₁-C₆alkoxy,    fluoro-substituted-C₁-C₆alkyl, fluoro-substituted-C₁-C₆alkoxy, N₃,    NH₂ and CN;-   R³ is selected from OH, NH₂, H and NHC(O)C₁-C₆alkyl;-   Z is selected from:

wherein,

-   R⁴ is selected from H, C₁-C₆alkyl and    hydroxy-substituted-C₁-C₆alkyl;-   One of R⁵ and R⁶ is hydrogen and the other is selected from H, OH    and F and one of-   R^(5′) and R^(6′) is hydrogen and the other is selected from H, OH    and F or R⁵ and R⁶ or R^(5′) and R^(6′) together may be ═O or ═CH₂;-   R⁷ is selected from H, F and OH;-   R⁸ is selected from H, C(O)C₁-C₆alkyl, P(O)(OH)₂, P(O)(OC₁-C₆alkyl)₂    and P(O)(OC₁-C₆alkyl)OH;-   R⁹ is selected from H, N₃, CN, C₁-C₆alkyl; and-   X—Y is selected from —CH₂—O—, —O—CH₂— and —S—CH₂—.

All of the compounds of Formula I have more than one asymmetric centre.Where the compounds according to the invention possess more than oneasymmetric centre, they may exist as diastereomers. It is to beunderstood that all such isomers and mixtures thereof in any proportionare encompassed within the scope of the present invention. In suitableembodiments of the invention, the stereochemistry is that found in thenatural form of uridine as depicted above. It is to be understood thatwhile, the relative stereochemistry of the compounds of Formula I issuitably as shown above, such compounds of Formula I may also containcertain amounts (e.g. less than 20%, preferably less than 10%, morepreferably less than 5%) of compounds of Formula I having alternatestereochemistry.

In further embodiments, the present invention includes a use of acompound selected from a compound of Formula I as defined above, andpharmaceutically acceptable salts, solvates, and prodrugs thereof, forthe prevention or treatment of viral and/or bacterial infections as wellas a use of a compound selected from a compound of Formula I as definedabove, and pharmaceutically acceptable salts, solvates, and prodrugsthereof, for the preparation of a medicament for the prevention ortreatment of viral and/or bacterial infections.

The present invention further includes a method of preventing ortreating an infection of one or more bacteria and/or one or more viruesin a subject comprising administering to the subject an anti-viraleffective amount and/or an antibacterial effective amount of a compoundselected from a compound of Formula I as defined above, andpharmaceutically acceptable salts, solvates, and prodrugs thereof.

The present invention also includes a use of a compound of Formula I asdefined above, and pharmaceutically acceptable salts, solvates, andprodrugs thereof, to prevent or treat an infection of a virus and/orbacterium in a subject as well as a use of a compound of Formula I asdefined above, and pharmaceutically acceptable salts, solvates, andprodrugs thereof, to prepare a medicament to prevent or treat aninfection of a virus and/or bacterium in a subject

According to another aspect of the present invention, there is includeda pharmaceutical composition for the treatment or prevention of viraland/or bacterial infections comprising an anti-viral effective amountand/or an antibacterial effective amount of a compound selected from acompound of Formula I as defined above, and pharmaceutically acceptablesalts, solvates, and prodrugs thereof, and a pharmaceutically acceptablecarrier or diluent.

The compounds of the invention are suitably formulated intopharmaceutical compositions for administration to human subjects in abiologically compatible form suitable for administration in vivo.

The compositions containing the compounds of the invention can beprepared by known methods for the preparation of pharmaceuticallyacceptable compositions which can be administered to subjects, such thatan effective quantity of the active substance is combined in a mixturewith a pharmaceutically acceptable vehicle. Suitable vehicles aredescribed, for example, in Remington's Pharmaceutical Sciences(2003—20th edition) and in The United States Pharmacopeia: The NationalFormulary (USP 24 NF19) published in 1999. On this basis, thecompositions include, albeit not exclusively, solutions of thesubstances in association with one or more pharmaceutically acceptablevehicles or diluents, and contained in buffered solutions with asuitable pH and iso-osmotic with the physiological fluids.

The compounds of Formula I may be used pharmaceutically in the form ofthe free base, in the form of salts, solvates and as hydrates. All formsare within the scope of the invention. Acid and basic addition salts maybe formed with the compounds of the invention for use as sources of thefree base form even if the particular salt per se is desired only as anintermediate product as, for example, when the salt is formed only forthe purposes of purification and identification. All salts that can beformed with the compounds of the invention are therefore within thescope of the present invention.

In accordance with the methods of the invention, the described compoundsof the invention, may be administered to a patient in a variety of formsdepending on the selected route of administration, as will be understoodby those skilled in the art. The compounds of the invention may beadministered, for example, by oral, parenteral, buccal, sublingual,nasal, rectal, patch, pump or transdermal administration and thepharmaceutical compositions formulated accordingly. Parenteraladministration includes intravenous, intraperitoneal, subcutaneous,intramuscular, transepithelial, nasal, intrapulmonary, intrathecal,rectal and topical modes of administration. Parenteral administrationmay be by continuous infusion over a selected period of time.

A compound of the invention may be orally administered, for example,with an inert diluent or with an assimilable edible carrier, or it maybe enclosed in hard or soft shell gelatin capsules, or it may becompressed into tablets, or it may be incorporated directly with thefood of the diet. For oral therapeutic administration, the compound ofthe invention may be incorporated with excipient and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like.

A compound of the invention may also be administered parenterally.Solutions of a compound of the invention can be prepared in watersuitably mixed with a surfactant such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, DMSO and mixtures thereof with or without alcohol, and in oils.Under ordinary conditions of storage and use, these preparations containa preservative to prevent the growth of microorganisms. A person skilledin the art would know how to prepare suitable formulations. Conventionalprocedures and ingredients for the selection and preparation of suitableformulations are described, for example, in Remington's PharmaceuticalSciences (2003—20th edition) and in The United States Pharmacopeia: TheNational Formulary (USP 24 NF19) published in 1999.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersion and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists.

Compositions for nasal administration may conveniently be formulated asaerosols, drops, gels and powders. Aerosol formulations typicallycomprise a solution or fine suspension of the active substance in aphysiologically acceptable aqueous or non-aqueous solvent and areusually presented in single or multidose quantities in sterile form in asealed container, which can take the form of a cartridge or refill foruse with an atomizing device. Alternatively, the sealed container may bea unitary dispensing device such as a single dose nasal inhaler or anaerosol dispenser fitted with a metering valve which is intended fordisposal after use. Where the dosage form comprises an aerosoldispenser, it will contain a propellant which can be a compressed gassuch as compressed air or an organic propellant such asfluorochlorohydrocarbon. The aerosol dosage forms can also take the formof a pump-atomizer.

Compositions suitable for buccal or sublingual administration includetablets, lozenges, and pastilles, wherein the active ingredient isformulated with a carrier such as sugar, acacia, tragacanth, or gelatinand glycerine. Compositions for rectal administration are convenientlyin the form of suppositories containing a conventional suppository basesuch as cocoa butter.

The compounds of the invention, may be administered to an animal aloneor in combination with pharmaceutically acceptable carriers, as notedabove, the proportion of which is determined by the solubility andchemical nature of the compound, chosen route of administration andstandard pharmaceutical practice.

The compounds of the invention, can be used alone or contemporaneouslywith other agents that inhibit ODCase activity or in combination withother types of treatment (which may or may not modulate ODCase) fortreating viral and/or bacterial infections.

III. Methods of Preparing Compounds of the Invention

In accordance with another aspect of the present invention, thecompounds of the invention can be prepared by processes analogous tothose established in the art. In particular, reactions forfunctionalizing the 5 and/or 6 position of a uracil, cytosine or thyminering are well known. For example, treatment of uracil, cytosine orthymine with a strong base, such as an alkyl lithium or lithiumdiisopropyl amide, at reduced temperatures, such at about −60° C. toabout −90° C., followed by reaction with a reagent of the Formula R¹-LG,where R¹ is as defined in Formula I and LG is a suitable leaving group,such as halo, provides a compound substituted at the 6-position of thepyrimidine ring with R¹. Compounds substituted with a suitable leavinggroup, such as I or Br, at the 5-position of the pyrimidine ring ofuracil or cytosine are commercially available or are known in the art.These compounds may be converted to their corresponding anions atreduced temperatures, such at about −60° C. to about −90° C., andreacted with a reagent of the Formula R²-LG, wherein R² is as defined inFormula I and LG is a suitable leaving group, such as halo to provide acompound substituted at the 5-position of the pyrimidine ring with R².Conversion of various R¹ groups into other R¹ groups can be done usingstandard chemistries known to those skilled in the art.

Pyrimidine compounds may be reacted with a reagent of the formula Z-LG,wherein Z is as defined in Formula I and LG is a suitable leaving group,under standard conditions to provide nucleosides of Formula I orprecursors to Formula I. Such reactions would be well known to thoseskilled in the art. Substitution of the appropriate R¹, R² and/or R³groups on the pyrimidine ring may be done before or after the couplingof the pyrimidine ring with Z.

Pyrimidine compounds and reagents of the Formula Z-LG are commerciallyavailable or may be prepared using methods known in the art. Acylationor addition of the phosphate group on to the 5′ position of thenucleoside may be performed using known reactions.

In some cases the chemistries outlined above may have to be modified,for instance by use of protective groups, to prevent side reactions dueto reactive groups, such as reactive groups attached as substituents.This may be achieved by means of conventional protecting groups, forexample as described in “Protective Groups in Organic Chemistry” McOmie,J. F. W. Ed., Plenum Press, 1973 and in Greene, T. W. and Wuts, P. G.M., “Protective Groups in Organic Synthesis”, John Wiley & Sons, 3^(rd)Edition, 1999.

The formation of a desired compound salt is achieved using standardtechniques. For example, the neutral compound is treated with an acid orbase in a suitable solvent and the formed salt is isolated byfiltration, extraction or any other suitable method.

The formation of solvates of the compounds of the invention will varydepending on the compound and the solvate. In general, solvates areformed by dissolving the compound in the appropriate solvent andisolating the solvate by cooling or using an antisolvent. The solvate istypically dried or azeotroped under ambient conditions.

Prodrugs of the compounds of Formula I may be, for example, conventionalesters formed with available hydroxy, thiol, amino or carboxyl group.For example, available hydroxy or amino groups may be acylated using anactivated acid in the presence of a base, and optionally, in inertsolvent (e.g. an acid chloride in pyridine). Some common esters whichhave been utilized as prodrugs are phenyl esters, aliphatic (C₈-C₂₄)esters, acyloxymethyl esters, carbamates and amino acid esters.

The present invention includes radiolabeled forms of the compounds ofthe invention, for example, compounds of the invention labeled byincorporation within the structure ³H, ¹¹C or ¹⁴C or a radioactivehalogen such as ¹²⁵I and ¹⁸F. A radiolabeled compound of the inventionmay be prepared using standard methods known in the art. For example,tritium may be incorporated into a compound of the invention usingstandard techniques, for example by hydrogenation of a suitableprecursor to a compound of the invention using tritium gas and acatalyst. Alternatively, a compound of the invention containingradioactive iodo may be prepared from the corresponding trialkyltin(suitably trimethyltin) derivative using standard iodination conditions,such as [¹²⁵I] sodium iodide in the presence of chloramine-T in asuitable solvent, such as dimethylformamide. The trialkyltin compoundmay be prepared from the corresponding non-radioactive halo, suitablyiodo, compound using standard palladium-catalyzed stannylationconditions, for example hexamethylditin in the presence oftetrakis(triphenylphosphine) palladium (0) in an inert solvent, such asdioxane, and at elevated temperatures, suitably 50-100° C. Further, acompound of the invention containing a radioactive fluorine may beprepared, for example, by reaction of K[¹⁸F]/K222 with a suitableprecursor compound, such as a compound of Formula I comprising asuitable leaving group, for example a tosyl group, that may be displacedwith the ¹⁸F anion.

The following non-limiting examples are illustrative of the presentinvention:

VI. Examples Example 1 Synthesis of 6-iodo uridine (Ia) and6-iodo-uridine-5′-O-monophosphate (Ib)

Compounds Ia and Ib were synthesized from uridine. Introduction of theiodo moiety at the C-6 position of protected uridine was achieved usinglithium diisopropylamide followed by treatment with iodine.^(xxxiv)Deprotection with TFA followed gave compound Ia, and the subsequentphosphorylation with phosphorus oxychloride afforded the mononucleotideIjb^(xxxv,xxxvi,xxxvii) Then, the compound Ib was transformed into itsammonium salt by neutralization with 0.5 M NH₄OH solution at 0° C. andfreeze-dried to get the ammonium salt as a powder.

(a) 5′-O-(t-Butyldimethylsilyl)-2′,3′-O-isopropylidene uridine. Astirred suspension of uridine (1 g, 4.1 mmol) in anhydrous acetone (50mL) was treated with H₂SO₄ (0.5 mL) drop wise at room temperature andthe resulting mixture was stirred for an additional hour. The reactionwas then neutralized with Et₃N and was concentrated. The crude mixturewas purified by column chromatography (5-8% MeOH:CHCl₃) to afford2′,3′-O-isopropylidene uridine (1.15 g, quant.) as a white solid. ¹H NMR(CDCl₃) d ppm 1.36 (s, 3H, —CH₃), 1.57 (s, 3H, —CH₃), 3.80 (dd, 1H,H-5′), 3.91 (dd, 1H, H-5″), 4.26-4.30 (m, 1H, H-4′), 4.95 (dd, 1H,H-3′), 5.02 (dd, 1H, H-1-2′) 5.56 (d, 1H, H-1′), 5.72 (d, 1H, H-5), 7.36(d, 1H, H-6).

A stirred solution of 2′,3′-O-isopropylidene uridine (0.2 g, 0.7 mmol)in anhydrous CH₂Cl₂ (3 mL) was treated with imidazole (0.095 g, 1.4mmol) and TBDMSiCl (0.105 g, 0.7 mmol) at 0° C. The reaction mixture wasbrought to room temperature and stirred for an additional hour. Thesolvent was evaporated under vacuum and the crude was dissolved in ethylacetate (30 mL), washed with water (15 mL), brine (15 mL) and dried(Na₂SO₄). Evaporation of the solvent and purification of the crude bycolumn chromatography (5% MeOH in CHCl₃) yielded5′-O-(t-butyldimethylsilyl)-2′,3′-O-isopropylidene uridine (0.27 mg, 96%yield) as a foam: ¹H NMR (CDCl₃) d ppm 0.10 (s, 6H, CH₃), 0.90 (s, 9H,CH₃), 1.36 (s, 3H, CH₃) 1.59 (s, 3H, CH₃), 3.79 (dd, 1H, H5′), 3.92 (dd,1H, H-5″), 4.30-4.33 (m, 1H, H-4′), 4.67 (dd, 1H, H-3′), 4.75 (dd, 1H,H-2′), 5.66 (d, 1H, H-5), 5.96 (dd, 1H, H-1′), 7.68 (d, 1H, H-6), 8.47(brs, H-1, —NH).

(b) 5′-O-(t-Butyldimethylsilyl)-6-iodo-2′,3′-O-isopropylidene uridine. Astirred solution of LDA (0.62 mL, 1.3 mmol, 2.0 M solution in THF) inanhydrous THF (2 mL) was treated with5′-O-(t-butyldimethylsilyl)-2′,3′-O-isopropylidene uridine (0.25 g, 0.6mmol) dissolved in 1.5 mL anhydrous THF, at −78° C. After stirring for 1h, iodine (0.16 g, 0.6 mmol) in anhydrous THF (2 mL) was added and themixture was stirred for an additional 5 h at the same temperature. Thereaction was quenched with AcOH (0.3 mL), then brought to roomtemperature and dissolved in ethyl acetate (25 mL). The organic layerwas washed with saturated NaHCO₃ solution (10 mL), 5% Na₂S₂O₃ solution(10 mL), brine (10 mL) and dried (Na₂SO₄). Evaporation of the solventand purification of the crude by column chromatography (hexanes-ethylacetate, 70:30) gave5′-O-(t-butyldimethylsilyl)-6-iodo-2′,3′-O-isopropylidene uridine (0.224g, 68%) as a yellow foam: ¹H NMR (CDCl₃) d ppm 0.06 (s, 6H, CH₃), 0.89(s, 9H, 3CH₃), 1.35 (s, 3H, CH₃) 1.56 (s, 3H, CH₃), 3.76-3.86 (m, 2H,H5′, H-5″), 4.15-4.20 (m, 1H, H-4′), 4.81 (dd, 1H, J=4.2, 6.3 Hz, H-3′),5.18 (dd, 1H, J=2.0, 6.3 Hz, H-2′), 6.09 (s, 1H, H-5), 6.45 (dd, 1H,J=2.0 Hz, H-1′), 8.78 (brs, 1H, NH).

(c) 6-Iodo-uridine (Ia). A stirred solution of5′-O-(t-butyldimethylsilyl)-6-iodo-2′,3′-O-isopropylidene uridine (0.300g, 0.572 mmol) was treated with 50% aqueous TFA (3 mL) at 0° C., broughtto room temperature and stirred for 2 h in the dark. Evaporation of thesolvent and purification of the crude by column chromatography (10-15%EtOH in CHCl₃) afforded 6-iodo uridine Ia (0.182 g, 0.49 mmol, 86%) as alight brown solid. UV (H₂O): λ_(max)=268 nm (e=8975); ¹H NMR (D₂O) δ ppm3.77 (dd, 1H, H-5′), 3.91 (dd, 1H, H-5″), 3.978-4.032 (m, 1H, H-4′),4.43 (t, 1H, H-3′), 4.84 (dd, 1H, H-2′), 6.06 (d, 1H, H-1′), 6.67 (s,1H, H-5). HRMS (ESI) calculated for C₉H₁₁N₂O₆NaI (M+Na⁺) 392.9554, found392.9565.

(d) 6-Iodo uridine-5′-O-monophosphate (Ib). A stirred solution of H₂O(0.034 g, 1.89 mmol) and POCl₃ (0.28 mL, 2.97 mmol) in anhydrousacetonitrile (3 mL) was treated with pyridine (0.261 mL, 3.24 mmol) at0° C. and stirred for 10 min. 6-Iodo uridine (0.250 g, 0.67 mmol) wasadded and the mixture was stirred for an additional 5 h at 0° C. Thereaction mixture was then quenched with 25 mL of cold water andcontinued stirring for an additional hour. The evaporation of thesolvent and purification of the crude by column chromatography (Dowexion-exchange basic resin, 0.1 M formic acid) afforded 6-iodouridine-5′-O-monophosphate (Ib) (0.207 g, 68%) as a syrup. UV (H₂O):λ_(max)=267 nm (e=2890); ¹H NMR (D₂O) δ ppm 3.78 (dd, 1H, H-5′), 3.91(dd, 1H, H-5″), 3.98-4.03 (m, 1H, H-4′), 4.43 (t, H-3′), 4.84 (dd, 1H,H-2′), 6.05 (d, 1H, H-1′), 6.67 (s, 1H, H-5). ³¹P NMR (D₂O) δ ppm 2.214.HRMS (ESI, negative) calculated for C₉H₁₁N₂O₉PI (M⁻) 448.9252, found448.9263.

Example 2 Synthesis of compounds Ic and Id

Introduction of the iodo moiety at the C-6 position of fully protecteduridine was achieved through LDA and iodine, and further substitution ofthe iodo by the azido group produced the 6-azido derivative shown in theabove scheme.^(xxxviii) Deprotection of the isopropylidene andt-butyldimethylsilyl groups using trifluoroacetic acid yielded6-azido-uridine Ic. Monophosphorylation of Id with phosphorusoxychloride to afford its mononucleotide followed by the reduction ofthe azido group with Pd/C gave the compound6-amino-uridine-5′-O-monophosphate Ic in good yield.^(xxxix,xl,xli)

(a) 6-Azido-5′-O-(t-butyldimethylsilyl)-2′,3′-O-isopropylidene uridine.5′-O-(t-Butyldimethylsilyl)-2′,3′-O-isopropylidene-6-iodo uridine (0.25g, 0.48 mmol) was dissolved in dry DMF (3 mL) and NaN₃ (0.034 g, 0.53mmol) was added. The reaction mixture was stirred at room temperaturefor 1 hr in the dark. Organic solvent was evaporated under vacuum andthe crude was dissolved in ethyl acetate (15 mL), washed with brine anddried (Na₂SO₄). Organic layers were evaporated and the crude waspurified by silica gel column chromatography (1% EtOH:CHCl₃).Purification of the compound and solvent evaporation were performed inthe dark to yield the title compound6-azido-5′-O-(t-butyldimethylsilyl)-2′,3′-O-isopropylidene uridine (0.19g, 0.44 mmol) in 92% yield as a light brown solid. ¹H NMR (CDCl₃) d 0.06(s, 6H), 0.89 (s, 9H), 1.34 (s, 3H) 1.54 (s, 3H), 3.74-3.85 (m, 2H),4.08-4.15 (m, 1H), 4.80 (dd, 1H, J=4.8, 6.3 Hz), 5.14 (dd, 1H, J=1.5,6.3 Hz), 5.50 (s, 1H), 6.09 (dd, 1H, J=1.5 Hz), 9.12 (brs, 1H).

(b) 6-Azido uridine (Ic). A stirred solution of6-azido-5′-O-(t-butyldimethylsilyl)-2′,3′-O-isopropylidene uridine(0.300 g, 0.683 mmol) was treated with 50% aqueous trifluoroacetic acid(3 mL) at 0° C. The reaction mixture was then brought to r.t. and wasstirred for an additional hour. Evaporation of the solvent andpurification of the crude by column chromatography (10-15% EtOH inCHCl₃) gave 6-azido uridine Ic (0.17 g, 0.61 mmol) in 89% yield as alight brown solid. UV (H₂O): λ_(max)=285 nm; ¹H NMR (D₂O) δ 3.77 (dd,1H, J=5.4, 12.0 Hz), 3.89-4.00 (m, 2H), 4.43 (t, J=6.9 Hz 1H), 4.77 (dd,1H, J=3.6, 6.9 Hz), 5.76 (s, 1H), 6.07 (d, 1H, J=3.6 Hz). HRMS (ESI)Calculated for C₉H₁₁N₅O₆Na (M+Na⁺) 308.0601, found 308.0597.

(c) 6-Azido uridine-5′-O-monophosphate (Id). A stirred solution of water(0.03 g, 1.89 mmol) and POCl₃ (0.28 mL, 2.97 mmol) in anhydrousacetonitrile (3 mL) was treated with pyridine (0.26 mL, 3.24 mmol) at 0°C. and stirred for 10 min. 6-Azido uridine Ic was added (0.25 g, 0.68mmol) and the mixture was stirred for an additional 5 hr at 0° C. Thereaction mixture was quenched with 25 mL of cold water and the stirringwas continued for another hour. Evaporation of the solvent andpurification of the crude by column chromatography (Dowex ion-exchangebasic resin, 0.1 M formic acid) gave the mononucleotide Id (0.23 g, 0.63mmol) in 60% yield as syrup. UV (H₂O) λ_(max)=283 nm; ¹H NMR (D₂O) δ3.78-3.85 (m, 1H), 3.89-4.00 (m, 2H), 4.34 (t, J=6.9 Hz 1H), 4.80 (m,1H), 5.73 (s, 1H), 6.04 (brs, 1H). ³¹P NMR (D₂O) δ ppm 2.47. HRMS (ESLnegative) Calculated for C₉H₁₁N₅O₉P (M⁻) 364.0299, found 364.0307.

Example 3 Anti-Viral Activity

Molecules containing the core structure, formula I, with specificsubstitutions at C-6 position (R₁) of the pyrimidine moiety are eithernoncovalent or covalent inhibitors of orotidine monophosphatedecarboxylase (ODCase). The molecular structures listed above alsoinclude, but are not limited to, all chemically-reasonable tautomericforms of the above structures as well as the prodrugs forms that releasethe above mentioned compounds and their tautomers. These molecules,described above, exhibit antiviral activities and protect the cells fromviral infections. Such molecules can be used in the treatment of viralinfections either alone or in combination with other methods oftreatment.

Selected compounds of Formula I were incubated with MDCK cells (forinfluenza virus A/WSN/33) and L2 cells (for MHV-1 infection—a mouse SARSlike corona virus representing a model for human SARS-like corona virus)for 5 hrs with 100 μM compounds. These compound-treated cells were theninfected with the appropriate virus with a dose equivalent to kill 100%cells within 24 h. Results for compounds Ia and Ib are depicted in FIGS.4 and 5. Selected compounds of Formula I were evaluated for their effecton the human primary blood cells for any indications of toxicity.Compound Ia did not inhibit primary cells significantly.

Example 4 Antibacterial Activity

ODCase gene, pyrF, was obtained from the complete genomes of therespective bacteria through ATCC. The pyrF gene was amplified from thegenome of S. aureus M252 using two synthetic primers: Dir5′-CCGGAATTCATGATGAAAGATTTACCAATTATTGCATTAG [SEQ ID NO: 1] and Rev5′-CGGAAGCTTTTATACTAACCAACTTTCTTTAATTTTATGATAACTTTCG [SEQ ID NO: 2].

The pET expression system was used to express ODCase from pyrF. Thecloning strategy can use any generic strategies, herein a doubledigestion of pET24a(+) expression vector and pyrF separately using EcoRIand HindIII restriction enzyme was used to produce digestion productswith sticky ends. The sticky digestion products were then ligatedtogether producing pyrF::pET24a(+) cloned DNA. pET24a(+) vector wasisolated from pET24a(+)/NovaBlue stock cells which were grown overnightat 37° C. in LB growth medium containing 30 mg/mL kanamyocin. pET24a(+)vector was isolated from the NovaBlue cells via QIAprep spin Miniprepkit using microcentrifuge Protocol. The isolated vector was doubledigested in 50 uL solution containing 1× EcoRI buffer, 1 uL EcoRI (20000 U/mL) and 0.5 uL HindIII (20 000 U/mL) producing sticky ends. Thedigested vector was separated from the restriction enzyme by gelpurification by QIAquick Gel Extraction Kit Protocol usingmicrocentrifuge and quantified on 1% agarose gel with 2-log ladder asstandard. In addition to quantifying DNA, 1% agarose gel was run withundigested vector to confirm that the vector was digested.

pyrF from the PCR mixture was also double digested in 50 uL solutionusing the same conditions described above. The separation of the pyrFfrom restriction enzymes was carried out by QIAquick Gel Extraction KitProtocol using Microcentrifuge, and the collected gene was quantified on1% agarose gel. The digested/purified pyrF was then ligated to thedigested/purified pET24a(+) using T4 Ligase. The ligation reaction wascarried out in 10 uL solution containing 70 ng of vector and mass ofgene that was 5× that of the vector. The reaction was incubated at 37°C. for 3 hours. 5 uL of the ligation mixture was then used to transformthe ligation product (pyrF::pET24a(+)) to NovaBlue cells and was platedonto Agar/LB/Kanamycin plates. The pyrF::pET24a(+) vector was isolatedfrom NovaBlue cells and used to transform BL21(DE3) cells. To induceODCase expression, pyrF::pET24a(+)/BL21(DE3) was grown in 1 L TB growthmedium containing 30 mg/mL Kanamyocin. The cells were grown at 37° C.until OD₆₀₀=0.6. Once OD₆₀₀=0.6, the cells were induced by the additionof IPTG to a final concentration of 0.3 mM. The induced cells wereincubated for 16 hours at 25° C. The cells were harvested bycentrifugation and the collected pellet was stored at −80° C. until use.Harvested cells were dissolved in 30 mL of 20 mM Tris (pH 7.8) bufferand sonicated to release the proteins within the cell followed bycentrifugation to collect insoluble components, including membranes andproteins as a pellet. The pellet was discarded and the supernatant wasdirectly loaded to a 100 mL DEAE anion exchange column. Bound proteinwas eluted by an increasing sodium chloride gradient (0.1 to 0.4 Msodium chloride). Samples from the elution fraction were run on 15% SDSgel and stained with Coomassie Blue staining solution to determineprotein content and purity of the fractions. The proteins were thenloaded to a 320 mL Sephacryl S-200 HiPrep26-60 size exclusion column.The proteins were eluted using 1×PBS buffer at pH 7.2. The fractionscontaining the enzyme were concentrated and desalted by buffer exchangewith 20 mM Tris (pH 7.8).

ODCase from H. pylori and other bacterial strains can be obtained usingthe above general procedure. This enzyme is then used to evaluate ODCaseinhibitory activities of nucleotide derivatives.

Enzyme inhibition assays were performed using standard protocols (seefor example Poduch, E.; Bello, A. M.; Tang, S.; Fujihashi, M.; Pai, E.F.; Kotra, L. P. J. Med. Chem. 2006, 49, 4937-4935).

Antimicrobial activities are carried out using standard protocols withvarious dilutions of the inhibitors such as the broth dilution method,and MIC₅₀ and other relevant parameters are computed.

The K₁ for compound Ib against Staphylococcus aureus ODCase was lessthan 50 nM indicating that this compound is a potent antibacterial agentfor this microorganism.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety. Where a term in the present application is found to bedefined differently in a document incorporated herein by reference, thedefinition provided herein is to serve as the definition for the term.

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1. A method of treating or preventing viral and/or bacterial infectionscomprising administering to a subject in need thereof a anti-viraleffective amount and/or an antibacterial effective amount of compoundselected from a compound of Formula I, tautomers thereof andpharmaceutically acceptable salts, solvates, and prodrugs thereof:

wherein, R¹ is selected from I, Cl, Br, N₃ and NO₂; R² is selected fromH, halo, C₁-C₆alkyl, C₁-C₆alkoxy, fluoro-substituted-C₁-C₆alkyl,fluoro-substituted-C₁-C₆alkoxy, N₃, NH₂ and CN; R³ is selected from OH,NH₂, H and NHC(O)C₁-C₆alkyl; Z is selected from:

wherein, R⁴ is selected from H, C₁-C₆alkyl andhydroxy-substituted-C₁-C₆alkyl; One of R⁵ and R⁶ is hydrogen and theother is selected from H, OH and F and one of R^(5′) and R^(6′) ishydrogen and the other is selected from H, OH and F or R⁵ and R⁶ orR^(5′) and R^(6′) together may be ═O or ═CH₂; R⁷ is selected from H, Fand OH; R⁸ is selected from H, C(O)C₁-C₆alkyl, P(O)(OH)₂,P(O)(OC₁-C₆alkyl)₂ and P(O)(OC₁-C₆alkyl)OH, R⁹ is selected from H, N₃,CN, C₁-C₆alkyl; and X—Y is selected from —CH₂—O—, —O—CH₂— and —S—CH₂—.2. The method according to claim 1, wherein R¹ in the compounds ofFormula I is selected from I, Br and Cl.
 3. The method according toclaim 2, wherein R¹ in the compounds of Formula I is I.
 4. The methodaccording to claim 1, wherein R² in the compounds of Formula I is H. 5.The method according to claim 1, wherein R² in the compounds of FormulaI is F.
 6. The method according to claim 1, wherein R³ in the compoundsof Formula I is selected from OH and NH₂.
 7. The method according toclaim 6, wherein R³ in the compounds of Formula I is OH and the compoundof Formula I has the following tautomeric structure:


8. The method according to claim 1, wherein in the compounds of FormulaI, Z is Formula II.
 9. The method according to claim 1, wherein R⁴ inthe compounds of Formula I is H.
 10. The method according to claim 1,wherein, R⁵ and R^(5′) are both OH and R⁶ and R^(6′) are both H.
 11. Themethod according to claim 1, wherein R⁵ is H, R^(5′) is OH and R⁶ andR^(6′) are both H.
 12. The method according to claim 1, wherein R⁷ inthe compounds of Formula I is H or OH.
 13. The method according to claim1, wherein R⁸ in the compounds of Formula I is selected from H,C(O)C₁-C₄alkyl, P(O)(OH)₂, P(O)(OC₁-C₄alkyl)₂ and P(O)(OC₁-C₄alkyl)OH.14. The method according to claim 13, wherein R⁸ in the compounds ofFormula I is selected from H, C(O)CH₃, P(O)(OH)₂, P(O)(OCH₃)₂ andP(O)(OCH₃)OH.
 15. The method according to claim 14, wherein R⁸ in thecompounds of Formula I is selected from H, C(O)CH₃, and P(O)(OH)₂. 16.The method according to claim 1, wherein R⁹ in the compounds of FormulaI is H.
 17. The method according to claim 1, wherein X—Y is O—CH₂—. 18.The method according to claim 1, wherein the compound of Formula I hasthe following structure:


19. The method according to claim 1, wherein the viral infection is anRNA viral infection.
 20. The method according to claim 19, wherein theRNA viral infection is from a viral family selected from Flaviviridae,Bunyaviridae and Togaviridae, or is from a virus selected from hepatitisC virus (HCV), hepatitis B virus (HBV), herpes viruses, influenza virus,human immuno virus (HIV), polio virus, Coxsackie A and B viruses, Rhinovirus, small pox virus, Ebola virus, West Nile virus and coronavirus.21. The method according to claim 1, wherein the bacterial infection isfrom a bacterium selected from H. pylori, S. aureus, B. anthracis,Mycobacterial species (such as M. tuberculosis, M. leprae and M. avium),P. aueruginosa, Streptococcal species and Pneumocystis carinii.
 22. Amethod of treating or preventing viral infections comprisingadministering to a subject in need thereof an anti-viral effectiveamount of a compound selected from a compound of Formula I, tautomersthereof and pharmaceutically acceptable salts, solvates, and prodrugsthereof:

wherein, R¹ is selected from I, Br, C₁, N₃ and NO₂; R² is H; R³ isselected from OH, NH₂, H and NHC(O)C₁-C₆alkyl; Z is selected from:

wherein, R⁴ is selected from H, C₁-C₆alkyl andhydroxy-substituted-C₁-C₆alkyl; One of R⁵ and R⁶ is hydrogen and theother is selected from H, OH and F and one of R^(5′) and R^(6′) ishydrogen and the other is selected from H, OH and F or R⁵ and R⁶ orR^(5′) and R^(6′) together may be ═O or ═CH₂; R⁷ is selected from H, Fand OH; R⁸ is selected from H, C(O)C₁-C₆alkyl, P(O)(OH)₂,P(O)(OC₁-C₆alkyl)₂ and P(O)(OC₁-C₆alkyl)OH; R⁹ is selected from H, N₃,CN, C₁-C₆alkyl; and X—Y is selected from —CH₂—O—, —O—CH₂— and —S—CH₂—.23. The method according to claim 22, wherein the viral infection is anRNA viral infection.
 24. The method according to claim 23, wherein theRNA viral infection is from a viral family selected from Flaviviridae,Bunyaviridae and Togaviridae, or is from a virus selected from hepatitisC virus (HCV), hepatitis B virus (HBV), herpes viruses, influenza virus,human immuno virus (HIV), polio virus, Coxsackie A and B viruses, Rhinovirus, small pox virus, Ebola virus, West Nile virus and coronavirus.25. The method according to claim 22, wherein the compound of Formula Iis selected from: 6-iodo uridine; 6-iodo uridine-5′-O-monophosphate;6-iodo uridine 5′-acetate; 6-iodo 2′-deoxyuridine 5′-acetate, andpharmaceutically acceptable salts, solvates, and prodrugs thereof.
 26. Amethod of treating or preventing bacterial infections comprisingadministering to a subject in need thereof an antibacterial effectiveamount compound selected from a compound of Formula I, tautomers thereofand pharmaceutically acceptable salts, solvates, and prodrugs thereof:

wherein, R¹ is selected from I, Br, C₁, N₃ and NO₂; R² is selected fromH, halo, C₁-C₆alkyl, C₁-C₆alkoxy, fluoro-substituted-C₁-C₆alkyl,fluoro-substituted-C₁-C₆alkoxy, N₃, NH₂ and CN; R³ is selected from OH,NH₂, H and NHC(O)C₁-C₆alkyl; Z is selected from:

wherein, R⁴ is selected from H, C₁-C₆alkyl andhydroxy-substituted-C₁-C₆alkyl; One of R⁵ and R⁶ is hydrogen and theother is selected from H, OH and F and one of R^(5′) and R^(6′) ishydrogen and the other is selected from H, OH and F or R⁵ and R⁶ orR^(5′) and R^(6′) together may be ═O or ═CH₂; R⁷ is selected from H, Fand OH; R⁸ is selected from H, C(O)C₁-C₆alkyl, P(O)(OH)₂,P(O)(OC₁-C₆alkyl)₂ and P(O)(OC₁-C₆alkyl)OH; R⁹ is selected from H, N₃,CN, C₁-C₆alkyl; and X—Y is selected from —CH₂—O—, —O—CH₂— and —S—CH₂—.27. The method according claim 26, wherein the bacterial infection isfrom a bacterium selected from H. pylori, S. aureus, B. anthracis,Mycobacterial species (such as M. tuberculosis, M. leprae and M. avium),P. aueruginosa, Streptococcal species and Pneumocystis carinii.
 28. Themethod according to claim 26, wherein the compound of Formula I isselected from: 6-iodo uridine; 5-fluoro-6-iodo uridine; 6-iodouridine-5′-O-monophosphate; 5-fluoro-6-iodo uridine-5′-O-monophosphate;6-iodo uridine 5′-acetate; 5-fluoro-6-iodo uridine-5′-acetate; 6-iodo2′-deoxyuridine; 5-fluoro-6-iodo 2′-deoxyuridine; 6-iodo2′-deoxyuridine-5′-O-monophosphate; 5-fluoro-6-iodo2′-deoxyuridine-5′-O-monophosphate, and pharmaceutically acceptablesalts, solvates, and prodrugs thereof.